Determinants of translation elongation speed and ribosomal profiling biases in mouse embryonic stem cells

Abstract

Ribosomal profiling is a promising approach with increasing popularity for studying translation. This approach enables monitoring the ribosomal density along genes at a resolution of single nucleotides.In this study, we focused on ribosomal density profiles of mouse embryonic stem cells. Our analysis suggests, for the first time, that even in mammals such as M. musculus the elongation speed is significantly and directly affected by determinants of the coding sequence such as: 1) the adaptation of codons to the tRNA pool; 2) the local mRNA folding of the coding sequence; 3) the local charge of amino acids encoded in the codon sequence. In addition, our analyses suggest that in general, the translation velocity of ribosomes is slower at the beginning of the coding sequence and tends to increase downstream.Finally, a comparison of these data to the expected biophysical behavior of translation suggests that it suffers from some unknown biases. Specifically, the ribosomal flux measured on the experimental data increases along the coding sequence; however, according to any biophysical model of ribosomal movement lacking internal initiation sites, the flux is expected to remain constant or decrease. Thus, developing experimental and/or statistical methods for understanding, detecting and dealing with such biases is of high importance.

Figure 1. Estimating the SL point. A diagram outlining the methodology for estimating starting location points (SL points) demonstrated on the uc007gge.1 isoform.

The black line depicts the ribosomal baseline profile while the red line represents a depleted profile (created using harringtonine to halt initiation; cychlohexamide was applied after 120 seconds). The dotted gray line represents the baseline profile multiplied by a recovery factor of 0.5, to which the depleted profile is compared. The red dotted line represents the estimated SL point.

Figure 2. (A). Estimated position of the SL points (mean and standard deviation): = 4.3+/−2.6,  = 6+/−2.5. v = 5.2+/−1.2 (Wilcoxon test: p = 2.2*10⁻²⁶).

(B). and histograms. (C). Histogram of measure calculated on: 1) the experimental data (blue) (median value 0.82) 2) on simulated ribosomal densities of the analyzed isoforms for low/high/proportional initiation rates (green/red/black) and 3) ribosomal densities created using codons of equal translation efficiency for low/high initiation rates (magenta/teal). For the simulations, the obtained median values of the measures were 0.06/0.06/0.06/0.02/0.01, significantly lower than in the case of the experimental data (KS p-value <6.18*10⁻¹⁵³ in all cases). The inset shows the ratio for the simulative data only. (D). Histogram of the ratio calculated on real and simulative data. The median value of this measure for the real ribosomal profiles was 1.37, significantly higher than for the simulative data, which resulted in median values of 1/1.01/1.01/1/1.01 accordantly (KS p-value <5.67*10⁻²⁵⁰ in all cases).

Figure 3. (A). Ribosome read counts measures according to the biophysical model.

The green round shapes represent the ribosomes on the mRNA, which is depicted with a blue line. According to the biophysical model, segments of high ribosomal read counts are associated with regions more slowly translated (bottom graph). The orange double arrows represent the mRNA segments being translated in equal time intervals. (B). histogram calculated on real (blue) and simulated ribosomal profiles for low/high/proportional initiation rates (black/red/green) with zero noise level. The calculated median value of this measure is 88/2.46/2.39/2.38 accordantly.

Figure 4. (A). vs. measured on the real data (B). vs. measured on the simulative data for low/high/proportional initiation rates (black circles/red crosses/green dots) (C). Histogram of the flux ratio measured on the real (blue; median = 1.69) and on the simulative data created using low/high/proportional initiation rates (black/red/green; median = 1/1.01/1.01; KS test: p-value <9*10⁻⁹⁵).